Analyze selection data using soluble Ephrin-B2 or -B3¶

In [1]:
# this cell is tagged as parameters for `papermill` parameterization
# input configs
altair_config = None
nipah_config = None

# input files
entropy_file = None
func_scores_E2_file = None
binding_E2_file = None
func_scores_E3_file = None
binding_E3_file = None

# output files
filtered_E2_binding_data = None
filtered_E3_binding_data = None
filtered_E2_binding_low_effect = None
filtered_E3_binding_low_effect = None

# output images
entry_binding_combined_corr_plot = None
entry_binding_combined_corr_plot_agg = None
E2_E3_correlation = None
E2_E3_correlation_site = None
combined_E2_E3_site_corr = None
binding_by_site_plot = None
entry_binding_corr_heatmap = None
binding_corr_heatmap = None
binding_region_boxplot_plot = None
binding_region_bubble_plot = None
max_binding_in_stalk = None
max_binding_in_contact = None
In [2]:
# Parameters
nipah_config = "nipah_config.yaml"
altair_config = "data/custom_analyses_data/theme.py"
entropy_file = "results/entropy/entropy.csv"
func_scores_E2_file = "results/func_effects/averages/CHO_EFNB2_low_func_effects.csv"
binding_E2_file = "results/receptor_affinity/averages/EFNB2_monomeric_mut_effect.csv"
func_scores_E3_file = "results/func_effects/averages/CHO_EFNB3_low_func_effects.csv"
binding_E3_file = "results/receptor_affinity/averages/EFNB3_dimeric_mut_effect.csv"
filtered_E2_binding_data = "results/filtered_data/E2_binding_filtered.csv"
filtered_E3_binding_data = "results/filtered_data/E3_binding_filtered.csv"
filtered_E2_binding_low_effect = (
    "results/filtered_data/E2_binding_low_effect_filter.csv"
)
filtered_E3_binding_low_effect = (
    "results/filtered_data/E3_binding_low_effect_filter.csv"
)
entry_binding_combined_corr_plot = (
    "results/images/entry_binding_combined_corr_plot.html"
)
entry_binding_combined_corr_plot_agg = (
    "results/images/entry_binding_combined_corr_plot_agg.html"
)
E2_E3_correlation = "results/images/E2_E3_correlation.html"
E2_E3_correlation_site = "results/images/E2_E3_correlation_site.html"
combined_E2_E3_site_corr = "results/images/combined_E2_E3_site_corr.html"
binding_by_site_plot = "results/images/binding_by_site_plot.html"
entry_binding_corr_heatmap = "results/images/entry_binding_corr_heatmap.html"
binding_corr_heatmap = "results/images/binding_corr_heatmap.html"
binding_region_boxplot_plot = "results/images/binding_region_boxplot_plot.html"
binding_region_bubble_plot = "results/images/binding_region_bubble_plot.html"
max_binding_in_contact = "results/images/max_binding_in_contact.html"
max_binding_in_stalk = "results/images/max_binding_in_stalk.html"
In [3]:
import math
import os
import re
import altair as alt
import numpy as np
import pandas as pd
import scipy.stats
import yaml
In [4]:
# allow more rows for Altair
_ = alt.data_transformers.disable_max_rows()

if (
    os.getcwd()
    == "/fh/fast/bloom_j/computational_notebooks/blarsen/2023/Nipah_Malaysia_RBP_DMS/"
):
    pass
    print("Already in correct directory")
else:
    os.chdir(
        "/fh/fast/bloom_j/computational_notebooks/blarsen/2023/Nipah_Malaysia_RBP_DMS/"
    )
    print("Setup in correct directory")
Setup in correct directory

hard paths for running in interactive mode¶

In [5]:
if nipah_config is None:
    ##hard paths in case don't want to run with snakemake
    print("loading hard paths")
    altair_config = "data/custom_analyses_data/theme.py"
    nipah_config = "nipah_config.yaml"
    entropy_file = "results/entropy/entropy.csv"

    # input files
    func_scores_E2_file = "results/func_effects/averages/CHO_EFNB2_low_func_effects.csv"
    binding_E2_file = (
        "results/receptor_affinity/averages/EFNB2_monomeric_mut_effect.csv"
    )
    func_scores_E3_file = "results/func_effects/averages/CHO_EFNB3_low_func_effects.csv"
    binding_E3_file = "results/receptor_affinity/averages/EFNB3_dimeric_mut_effect.csv"

    filtered_E2_binding_data = "results/filtered_data/E2_binding_filtered.csv"
    filtered_E3_binding_data = "results/filtered_data/E3_binding_filtered.csv"
    filtered_E2_binding_low_effect = (
        "results/filtered_data/E2_binding_low_effect_filter.csv"
    )
    filtered_E3_binding_low_effect = (
        "results/filtered_data/E3_binding_low_effect_filter.csv"
    )

Run config files to setup altair theme and config variables¶

In [6]:
if altair_config:
    with open(altair_config, "r") as file:
        exec(file.read())

with open(nipah_config) as f:
    config = yaml.safe_load(f)

Import the binding and entry data for EFNB2 and EFNB3¶

In [7]:
# import binding and entry data
e2 = pd.read_csv(binding_E2_file)
e2_func = pd.read_csv(func_scores_E2_file)
e3 = pd.read_csv(binding_E3_file)
e3_func = pd.read_csv(func_scores_E3_file)

Filter the data and save¶

In [8]:
def merge_func_binding_dfs(func, binding, name):
    df_int = pd.merge(
        binding,
        func,
        on=["site", "mutant", "wildtype"],
        suffixes=["_binding", "_cell_entry"],
        validate="one_to_one",
        how="outer",
    ).round(3)
    df = df_int.rename(
        columns={
            "Ephrin binding_mean": "binding_mean",
            "Ephrin binding_std": "binding_std",
            "Ephrin binding_median": "binding_median",
        }
    )

    # Only save relevant columns
    df = df[
        [
            "site",
            "wildtype",
            "mutant",
            "binding_median",
            "binding_std",
            "times_seen_binding",
            "effect",
            "effect_std",
            "times_seen_cell_entry",
            "frac_models",
        ]
    ]

    def filter_binding_data(df):
        df_filter = df[
            (df["mutant"] != "*")
            & (df["mutant"] != "-")
            & (df["site"] != 603)
            &
            # Filter cell entry parameters
            (df["effect"] >= config["min_func_effect_for_binding"])
            & (df["times_seen_cell_entry"] >= config["func_times_seen_cutoff"])
            & (df["effect_std"] <= config["func_std_cutoff"])
            &
            # Filter binding parameters
            (df["times_seen_binding"] >= config["min_times_seen_binding"])
            & (df["binding_std"] <= config["max_binding_std"])
            & (df["frac_models"] >= config["frac_models"])
        ]
        return df_filter

    df_filter = filter_binding_data(df)

    # For pulling out low effect mutants for heatmaps later. Find mutants below func effect cutoff, but still have ok times_seen and func_std.
    def store_filtered_info(df):
        df_low_filter = df[
            (df["mutant"] != "*")
            & (df["mutant"] != "-")
            & (df["site"] != 603)
            & (df["effect"] < config["min_func_effect_for_binding"])
            & (df["times_seen_cell_entry"] >= config["func_times_seen_cutoff"])
            & (df["effect_std"] <= config["func_std_cutoff"])
        ]
        return df_low_filter

    df_low_effect_filter = store_filtered_info(df)

    # Save the filtered data to .csv
    if name == "EFNB2":
        print(name)
        df_filter.to_csv(filtered_E2_binding_data, index=False)
        df_low_effect_filter.to_csv(filtered_E2_binding_low_effect, index=False)
    else:
        df_filter.to_csv(filtered_E3_binding_data, index=False)
        df_low_effect_filter.to_csv(filtered_E3_binding_low_effect, index=False)

    return df_filter, df_low_effect_filter


# Call filtering function
df_E2_filter, df_E2_filter_missing = merge_func_binding_dfs(e2_func, e2, "EFNB2")
df_E3_filter, df_E3_filter_missing = merge_func_binding_dfs(e3_func, e3, "EFNB3")

# Now that they are filtered, merge EFNB2 and EFNB3
df_binding_effect_merge = pd.merge(
    df_E2_filter,
    df_E3_filter,
    on=["site", "wildtype", "mutant"],
    suffixes=["_E2", "_E3"],
    how="outer",
)

# display stats
display(df_binding_effect_merge.describe().round(3))

# Make a concat df of E2/E3 data for plotting later
df_E2_filter["selection"] = "EFNB2"
df_E3_filter["selection"] = "EFNB3"
df_binding_effect_concat = pd.concat([df_E2_filter, df_E3_filter])
EFNB2
site binding_median_E2 binding_std_E2 times_seen_binding_E2 effect_E2 effect_std_E2 times_seen_cell_entry_E2 frac_models_E2 binding_median_E3 binding_std_E3 times_seen_binding_E3 effect_E3 effect_std_E3 times_seen_cell_entry_E3 frac_models_E3
count 7212.000 6684.000 6684.000 6684.000 6684.000 6684.000 6684.000 6684.000 6519.000 6519.000 6519.000 6519.000 6519.000 6519.000 6519.0
mean 343.684 -0.333 0.501 6.400 -0.061 0.368 7.699 0.998 -0.017 0.179 6.227 -0.039 0.388 6.804 1.0
std 148.183 1.095 0.321 3.203 0.495 0.191 4.383 0.022 0.271 0.169 3.053 0.474 0.186 3.584 0.0
min 71.000 -5.494 0.008 2.250 -1.500 0.010 2.000 0.750 -2.147 0.000 2.500 -1.500 0.033 2.000 1.0
25% 217.000 -0.416 0.273 4.500 -0.335 0.223 5.000 1.000 -0.150 0.062 4.500 -0.266 0.248 4.714 1.0
50% 342.000 -0.033 0.426 5.750 0.076 0.337 6.750 1.000 -0.012 0.135 5.500 0.094 0.356 6.000 1.0
75% 468.000 0.190 0.645 7.500 0.329 0.480 9.125 1.000 0.118 0.241 7.500 0.324 0.496 7.857 1.0
max 602.000 2.205 1.995 43.750 0.617 0.994 72.380 1.000 2.006 1.780 45.000 0.616 1.000 56.710 1.0
In [9]:
# What are the top 5 highest and lowest binding mutants for EFNB2 and EFNB3?
def find_highest_lowest(df, name):
    print(f"We are analyzing {name}\n")
    tmp_df = df.sort_values(by="binding_median")
    print("These are the lowest binding mutants detected:")
    display(tmp_df.head(5))

    tmp_df_high = df.sort_values(by="binding_median", ascending=False)
    print("\nThese are the highest binding mutants detected:\n")
    display(tmp_df_high.head(10))

    # What about mutants with positive entry scores?
    tmp_df = df[df["effect"] > 0].sort_values(by="binding_median")
    print("These are the lowest binding mutants detected with positive entry scores:")
    display(tmp_df.head(5))

    tmp_df_high = df[df["effect"] > 0].sort_values(by="binding_median", ascending=False)
    print(
        "\nThese are the highest binding mutants detected with positive entry scores:\n"
    )
    display(tmp_df_high.head(10))


find_highest_lowest(df_E2_filter, "EFNB2")
find_highest_lowest(df_E3_filter, "EFNB3")
We are analyzing EFNB2

These are the lowest binding mutants detected:
site wildtype mutant binding_median binding_std times_seen_binding effect effect_std times_seen_cell_entry frac_models selection
8285 501 E K -5.494 0.928 24.50 -0.712 0.606 32.00 1.0 EFNB2
8898 532 A V -5.180 1.140 16.25 -0.292 0.312 17.88 1.0 EFNB2
8072 490 Q K -4.871 1.081 8.50 -0.673 0.539 11.38 1.0 EFNB2
8070 490 Q H -4.864 0.862 8.25 -0.273 0.668 10.88 1.0 EFNB2
3188 236 R H -4.853 0.883 22.00 -1.217 0.335 29.38 1.0 EFNB2
These are the highest binding mutants detected:

site wildtype mutant binding_median binding_std times_seen_binding effect effect_std times_seen_cell_entry frac_models selection
5459 354 S R 2.205 1.165 4.25 -0.922 0.537 6.750 1.00 EFNB2
9830 580 I S 2.165 1.768 4.75 -0.750 0.692 6.375 1.00 EFNB2
9948 586 N T 2.096 0.578 5.25 -0.658 0.823 7.000 1.00 EFNB2
655 104 E T 2.094 0.755 4.50 -0.348 0.671 3.500 1.00 EFNB2
2696 211 G F 2.060 0.549 4.75 -0.753 0.426 5.125 1.00 EFNB2
2621 207 L I 2.042 1.531 4.00 -0.816 0.764 3.750 1.00 EFNB2
1298 138 I L 1.968 0.917 5.00 -1.114 0.244 4.750 1.00 EFNB2
2051 178 V H 1.938 0.730 3.25 -0.639 0.903 3.625 1.00 EFNB2
9307 553 S W 1.923 0.287 10.00 -0.396 0.429 13.250 1.00 EFNB2
4768 318 R P 1.922 1.433 3.00 -0.961 0.764 4.000 0.75 EFNB2
These are the lowest binding mutants detected with positive entry scores:
site wildtype mutant binding_median binding_std times_seen_binding effect effect_std times_seen_cell_entry frac_models selection
9980 588 I L -4.684 0.874 5.750 0.159 0.420 6.375 1.0 EFNB2
9402 558 A V -4.660 0.730 17.250 0.152 0.364 18.500 1.0 EFNB2
9369 557 N D -4.637 0.399 9.500 0.547 0.479 11.120 1.0 EFNB2
8146 494 P E -4.598 0.874 7.812 0.287 0.194 9.750 1.0 EFNB2
8773 526 L H -4.539 0.782 5.250 0.222 0.392 6.750 1.0 EFNB2
These are the highest binding mutants detected with positive entry scores:

site wildtype mutant binding_median binding_std times_seen_binding effect effect_std times_seen_cell_entry frac_models selection
9548 566 F H 1.388 0.847 4.25 0.186 0.554 4.375 1.0 EFNB2
7303 450 Q I 1.330 1.194 5.00 0.316 0.399 6.000 1.0 EFNB2
10034 591 K G 1.266 0.156 5.75 0.027 0.442 6.125 1.0 EFNB2
4540 306 N T 1.239 0.491 4.00 0.110 0.357 7.625 1.0 EFNB2
9513 564 N K 1.196 0.488 7.25 0.228 0.442 8.000 1.0 EFNB2
2034 177 G M 1.193 0.618 6.50 0.380 0.534 5.375 1.0 EFNB2
7184 444 I F 1.162 0.350 5.00 0.294 0.314 6.250 1.0 EFNB2
2519 202 L F 1.156 0.358 3.75 0.096 0.344 4.375 1.0 EFNB2
4594 309 Y P 1.148 1.065 3.00 0.389 0.521 2.875 1.0 EFNB2
9764 577 L I 1.115 0.101 5.75 0.041 0.398 5.500 1.0 EFNB2
We are analyzing EFNB3

These are the lowest binding mutants detected:
site wildtype mutant binding_median binding_std times_seen_binding effect effect_std times_seen_cell_entry frac_models selection
9221 555 D K -2.147 1.225 6.0 0.251 0.422 6.714 1.0 EFNB3
9227 555 D R -1.510 0.420 4.0 0.312 0.526 4.143 1.0 EFNB3
8739 530 Q L -1.447 0.477 3.5 -1.231 0.862 5.857 1.0 EFNB3
9761 583 T R -1.410 0.446 5.5 -0.870 0.485 6.286 1.0 EFNB3
9755 583 T K -1.247 0.113 5.0 -0.667 0.644 7.000 1.0 EFNB3
These are the highest binding mutants detected:

site wildtype mutant binding_median binding_std times_seen_binding effect effect_std times_seen_cell_entry frac_models selection
9866 589 R G 2.006 0.659 7.0 -1.197 0.576 8.286 1.0 EFNB3
9699 580 I M 1.337 0.534 5.5 -0.873 0.531 6.714 1.0 EFNB3
3777 270 V Q 1.329 1.028 5.0 -0.920 0.651 5.429 1.0 EFNB3
3719 267 M Q 1.291 1.371 4.5 -0.594 0.818 5.667 1.0 EFNB3
8010 492 Q L 1.261 0.067 5.5 0.548 0.183 5.143 1.0 EFNB3
1150 132 S Y 1.247 0.722 5.5 -1.024 0.573 6.000 1.0 EFNB3
9691 580 I C 1.241 0.524 4.0 -0.173 0.401 4.857 1.0 EFNB3
9871 589 R M 1.234 0.635 4.5 -0.726 0.342 5.429 1.0 EFNB3
9566 573 W M 1.215 1.373 4.0 -0.835 0.676 5.429 1.0 EFNB3
9853 588 I P 1.183 0.255 5.5 -0.607 0.382 5.429 1.0 EFNB3
These are the lowest binding mutants detected with positive entry scores:
site wildtype mutant binding_median binding_std times_seen_binding effect effect_std times_seen_cell_entry frac_models selection
9221 555 D K -2.147 1.225 6.0 0.251 0.422 6.714 1.0 EFNB3
9227 555 D R -1.510 0.420 4.0 0.312 0.526 4.143 1.0 EFNB3
9323 560 K S -1.022 0.438 5.5 0.019 0.446 5.833 1.0 EFNB3
8747 530 Q W -0.997 0.547 3.0 0.144 0.665 3.286 1.0 EFNB3
2784 218 T S -0.916 0.931 5.0 0.267 0.884 4.571 1.0 EFNB3
These are the highest binding mutants detected with positive entry scores:

site wildtype mutant binding_median binding_std times_seen_binding effect effect_std times_seen_cell_entry frac_models selection
8010 492 Q L 1.261 0.067 5.5 0.548 0.183 5.143 1.0 EFNB3
2637 211 G F 1.169 0.289 5.0 0.436 0.572 5.714 1.0 EFNB3
10031 598 P G 1.154 1.559 4.5 0.422 0.515 5.429 1.0 EFNB3
9192 553 S W 0.994 0.435 9.5 0.099 0.406 9.857 1.0 EFNB3
9745 582 D W 0.976 0.338 6.5 0.111 0.310 7.143 1.0 EFNB3
1663 161 S H 0.923 0.294 4.0 0.077 0.465 4.143 1.0 EFNB3
1818 169 R G 0.859 0.347 7.5 0.096 0.473 7.571 1.0 EFNB3
2977 228 Y R 0.810 0.113 5.0 0.173 0.346 5.714 1.0 EFNB3
1770 166 L W 0.807 0.348 6.5 0.358 0.341 6.429 1.0 EFNB3
1667 161 S M 0.782 0.376 4.5 0.063 0.370 5.857 1.0 EFNB3
In [10]:
# Compare E2 and E3 binders
def find_highest_lowest(df):
    df["binding_diff"] = (df["binding_median_E2"] - df["binding_median_E3"]).abs()
    print(
        "These are the mutants with the biggest difference between EFNB2 and EFNB3:\n"
    )
    display(df.sort_values(by="binding_diff", ascending=False).head(5))

    # calculate aggregate differences
    agg_df = (
        df.groupby("site")[["binding_median_E2", "binding_median_E3", "binding_diff"]]
        .max()
        .reset_index()
    )
    print("These are the sites with the biggest difference between EFNB2 and EFNB3:\n")
    display(agg_df.sort_values(by="binding_diff", ascending=False).head(5))


find_highest_lowest(df_binding_effect_merge)
# find_highest_lowest(df_E3_filter,'EFNB3')
These are the mutants with the biggest difference between EFNB2 and EFNB3:

site wildtype mutant binding_median_E2 binding_std_E2 times_seen_binding_E2 effect_E2 effect_std_E2 times_seen_cell_entry_E2 frac_models_E2 binding_median_E3 binding_std_E3 times_seen_binding_E3 effect_E3 effect_std_E3 times_seen_cell_entry_E3 frac_models_E3 binding_diff
5729 530 Q F -4.114 0.466 6.25 0.550 0.202 5.375 1.0 0.084 0.062 6.0 0.408 0.274 6.286 1.0 4.198
5258 490 Q W -4.519 1.005 3.75 0.407 0.378 4.750 1.0 -0.586 0.323 3.0 -0.130 0.458 3.286 1.0 3.933
5259 490 Q Y -4.089 0.900 5.50 -1.085 0.400 6.375 1.0 -0.461 0.574 5.5 -0.280 0.797 5.714 1.0 3.628
5263 491 S G -4.183 0.595 6.50 0.058 0.298 8.625 1.0 -0.562 0.047 5.5 -0.387 0.608 6.286 1.0 3.621
5283 492 Q K -3.484 0.549 11.00 0.430 0.232 11.620 1.0 0.006 0.077 10.0 0.388 0.240 11.570 1.0 3.490
These are the sites with the biggest difference between EFNB2 and EFNB3:

site binding_median_E2 binding_median_E3 binding_diff
441 530 0.628 0.405 4.198
405 490 -0.967 0.112 3.933
406 491 -0.799 0.645 3.621
407 492 0.362 1.261 3.490
162 239 -0.393 -0.041 3.418

Make plots showing correlation between binding and entry for EFNB2 and EFNB3¶

In [11]:
def plot_corr_binding_entry_updated(df, flag):
    variant_selector = alt.selection_point(
        on="mouseover", empty=False, fields=["site", "mutant"], value=0
    )
    variant_selector_agg = alt.selection_point(
        on="mouseover", empty=False, fields=["site"], value=0
    )
    slider = alt.binding_range(min=2, max=10, step=1, name="times seen")
    selector = alt.param(name="SelectorName", value=2, bind=slider)

    empty_chart = []
    for cell in list(df["selection"].unique()):
        tmp_df = df[df["selection"] == cell]
        if flag == True:
            agg_df = (
                tmp_df.groupby("site")[["binding_median", "effect"]].sum().reset_index()
            )
            chart = (
                alt.Chart(agg_df)
                .mark_point(stroke="black", filled=True)
                .encode(
                    x=alt.X(
                        "effect",
                        title=f"Summed {cell} Cell Entry",
                        axis=alt.Axis(grid=True),
                    ),
                    y=alt.Y(
                        "binding_median",
                        title=f"Summed {cell} Binding",
                        axis=alt.Axis(grid=True),
                    ),
                    opacity=alt.condition(
                        variant_selector_agg, alt.value(1), alt.value(0.2)
                    ),
                    size=alt.condition(
                        variant_selector_agg, alt.value(100), alt.value(50)
                    ),
                    strokeWidth=alt.condition(
                        variant_selector_agg, alt.value(1), alt.value(0)
                    ),
                    color=alt.condition(
                        variant_selector_agg, alt.value("orange"), alt.value("black")
                    ),
                    tooltip=["site", "binding_median", "effect"],
                )
                .add_params(variant_selector_agg)
            )

            empty_chart.append(chart)

        else:
            chart = (
                alt.Chart(tmp_df)
                .mark_point(stroke="black", filled=True)
                .encode(
                    x=alt.X(
                        "effect", title=f"{cell} Cell Entry", axis=alt.Axis(grid=True)
                    ),
                    y=alt.Y(
                        "binding_median",
                        title=f"{cell} Binding",
                        axis=alt.Axis(grid=True),
                    ),
                    opacity=alt.condition(
                        variant_selector, alt.value(1), alt.value(0.1)
                    ),
                    size=alt.condition(variant_selector, alt.value(50), alt.value(20)),
                    strokeWidth=alt.condition(
                        variant_selector, alt.value(1), alt.value(0)
                    ),
                    color=alt.condition(
                        variant_selector, alt.value("orange"), alt.value("black")
                    ),
                    tooltip=[
                        "site",
                        "wildtype",
                        "mutant",
                        "binding_median",
                        "times_seen_binding",
                        "effect",
                    ],
                )
                .add_params(variant_selector)
            )
            empty_chart.append(chart)

    combined_chart = alt.hconcat(
        *empty_chart, title=alt.Title("Correlation between binding and entry")
    )
    return combined_chart


entry_binding_corr_plot = plot_corr_binding_entry_updated(
    df_binding_effect_concat, False
)
entry_binding_corr_plot.display()
if entry_binding_combined_corr_plot is not None:
    entry_binding_corr_plot.save(entry_binding_combined_corr_plot)

entry_binding_corr_plot_agg = plot_corr_binding_entry_updated(
    df_binding_effect_concat, True
)
entry_binding_corr_plot_agg.display()
if entry_binding_combined_corr_plot is not None:
    entry_binding_corr_plot_agg.save(entry_binding_combined_corr_plot_agg)

Same plot as above, but slightly different format¶

In [12]:
def plot_entry_binding_corr_heatmap(df):
    empty_chart = []

    for cell in list(df["selection"].unique()):
        tmp_df = df[df["selection"] == cell]
        chart = (
            alt.Chart(tmp_df, title=f"{cell}")
            .mark_rect()
            .encode(
                x=alt.X(
                    "effect", title="Cell Entry", axis=alt.Axis(values=[-2, -1, 0, 1])
                ).bin(maxbins=60),
                y=alt.Y(
                    "binding_median",
                    title="Binding",
                    axis=alt.Axis(values=[-4, -2, 0, 2]),
                ).bin(maxbins=60),
                color=alt.Color("count()", title="Count").scale(scheme="greenblue"),
            )
        )
        empty_chart.append(chart)

    combined_chart = alt.hconcat(
        *empty_chart, title=alt.Title("Correlation between binding and entry")
    ).resolve_scale(y="shared", x="shared", color="shared")
    return combined_chart


entry_binding_corr_heat = plot_entry_binding_corr_heatmap(df_binding_effect_concat)
entry_binding_corr_heat.display()
if entry_binding_combined_corr_plot is not None:
    entry_binding_corr_heat.save(entry_binding_corr_heatmap)

Calculate some stats on binding¶

In [13]:
def overall_stats(df, effect, name):
    # Now group sites and find sites where all mutants are deleterious
    filtered_df = df.groupby("site").filter(lambda group: (group[effect] < -0.25).all())
    # Which sites are these?
    unique = filtered_df["site"].unique()
    # Convert unique to a Pandas Series
    unique_series = pd.Series(unique)

    # Find the common elements that are also contact sites
    unique_contact_bool = unique_series.isin(config["contact_sites"])
    # Filter and get the common elements
    common_elements = unique_series[unique_contact_bool]

    print(f"The dataset we are analyzing is: {name}\n")

    # Print the common elements
    print(
        f"Here are the contact sites that only have negative binding scores: {list(common_elements)}\n"
    )

    print(f"There are {len(unique)} sites with all negative binding score mutants\n")
    print(
        f"These are the sites with all negative binding score mutants: {list(unique)}\n"
    )

    # Now find sites with low and high binding (median)
    median_df = (
        df.groupby("site")["binding_median"]
        .max()
        .reset_index()
        .sort_values(by="binding_median", ascending=False)
    )
    print("These are the sites with the highest binding mutants:\n")
    display(median_df.head(5))

    # Now calculate mutant number
    total_mutants = df.shape[0]

    mutants_above_cutoff_tolerated = df[df["effect"] > 0]
    mutants_above_cutoff_tolerated = mutants_above_cutoff_tolerated[
        ["site", "effect", "binding_median", "wildtype", "mutant"]
    ]

    total_sites = df["site"].unique().shape[0]

    print(f"The total number of sites are: {total_sites}")


overall_stats(df_E2_filter, "binding_median", "EFNB2")
overall_stats(df_E3_filter, "binding_median", "EFNB3")
The dataset we are analyzing is: EFNB2

Here are the contact sites that only have negative binding scores: [238, 239, 242, 389, 488, 490, 491, 501, 504, 505, 531, 532, 533, 557, 579, 581, 588]

There are 43 sites with all negative binding score mutants

These are the sites with all negative binding score mutants: [116, 220, 236, 238, 239, 242, 243, 248, 346, 351, 352, 389, 390, 398, 399, 400, 435, 438, 441, 460, 467, 486, 487, 488, 490, 491, 494, 495, 497, 501, 504, 505, 526, 531, 532, 533, 557, 579, 581, 584, 585, 588, 590]

These are the sites with the highest binding mutants:

site binding_median
272 354 2.205
487 580 2.165
493 586 2.096
33 104 2.094
135 211 2.060
The total number of sites are: 510
The dataset we are analyzing is: EFNB3

Here are the contact sites that only have negative binding scores: [389, 488, 501, 531, 532]

There are 15 sites with all negative binding score mutants

These are the sites with all negative binding score mutants: [108, 140, 352, 389, 467, 486, 488, 494, 495, 497, 501, 510, 531, 532, 584]

These are the sites with the highest binding mutants:

site binding_median
490 589 2.006
482 580 1.337
189 270 1.329
186 267 1.291
405 492 1.261
The total number of sites are: 504

Find sites with opposite effects on binding¶

In [14]:
# find sites that are different
def find_biggest_differences(df):
    efnb2_good_efnb3_bad = df[
        (df["binding_median_E2"] > 0.5) & (df["binding_median_E3"] < -0.5)
    ].sort_values(by="binding_median_E2", ascending=False)
    display(efnb2_good_efnb3_bad)

    efnb2_bad_efnb3_good = df[
        (df["binding_median_E2"] < -0.5) & (df["binding_median_E3"] > 0.5)
    ].sort_values(by="binding_median_E3", ascending=False)
    display(efnb2_bad_efnb3_good)


find_biggest_differences(df_binding_effect_merge)
site wildtype mutant binding_median_E2 binding_std_E2 times_seen_binding_E2 effect_E2 effect_std_E2 times_seen_cell_entry_E2 frac_models_E2 binding_median_E3 binding_std_E3 times_seen_binding_E3 effect_E3 effect_std_E3 times_seen_cell_entry_E3 frac_models_E3 binding_diff
2555 303 P C 1.223 1.577 5.00 -0.745 0.590 5.000 1.0 -0.566 0.098 4.0 -0.527 0.594 4.857 1.0 1.789
5937 546 L H 1.161 1.145 5.25 -1.264 0.387 6.750 1.0 -0.543 0.665 3.5 -0.674 0.544 5.143 1.0 1.704
997 178 V T 0.796 0.726 3.75 -0.272 0.759 4.250 1.0 -0.513 0.600 3.0 -0.022 0.425 3.571 1.0 1.309
4692 447 S E 0.767 1.742 4.25 -1.116 0.812 4.750 1.0 -0.557 0.393 4.0 -0.078 0.325 4.571 1.0 1.324
1619 223 A M 0.736 1.538 3.25 -0.383 0.522 3.375 1.0 -0.561 0.206 3.0 -0.085 0.408 3.571 1.0 1.297
109 79 N S 0.692 1.652 3.00 -0.368 0.496 2.375 1.0 -0.646 0.260 3.0 -0.011 0.569 3.429 1.0 1.338
90 78 D I 0.521 0.634 5.25 -0.894 0.398 6.500 1.0 -0.566 0.755 5.0 -0.664 0.845 5.143 1.0 1.087
site wildtype mutant binding_median_E2 binding_std_E2 times_seen_binding_E2 effect_E2 effect_std_E2 times_seen_cell_entry_E2 frac_models_E2 binding_median_E3 binding_std_E3 times_seen_binding_E3 effect_E3 effect_std_E3 times_seen_cell_entry_E3 frac_models_E3 binding_diff
6512 588 I P -2.042 0.840 5.25 -0.276 0.300 4.750 1.0 1.183 0.255 5.5 -0.607 0.382 5.429 1.0 3.225
3107 338 R G -0.574 0.507 4.50 0.458 0.422 5.250 1.0 0.684 0.976 4.0 0.270 0.340 3.429 1.0 1.258
5260 491 S A -0.799 0.537 5.50 -0.441 0.806 6.250 1.0 0.645 0.010 6.5 0.232 0.358 6.143 1.0 1.444
5287 492 Q R -2.826 0.611 19.25 -0.003 0.272 23.120 1.0 0.644 0.054 20.0 0.549 0.119 20.570 1.0 3.470
4367 424 P A -0.502 0.276 3.50 0.306 0.322 3.625 1.0 0.592 0.229 2.5 0.329 0.188 2.857 1.0 1.094

Find correlations between EFNB2 and EFNB3 binding¶

In [15]:
def plot_entry_binding_corr(df):
    chart = (
        alt.Chart(df, title="Correlation Between Mutant Binding Scores")
        .mark_rect()
        .encode(
            x=alt.X(
                "binding_median_E2",
                title="EFNB2 binding",
                axis=alt.Axis(values=[-5, 0, 2]),
            ).bin(maxbins=40),
            y=alt.Y(
                "binding_median_E3",
                title="EFNB3 binding",
                axis=alt.Axis(values=[-2, 0, 2]),
            ).bin(maxbins=40),
            color=alt.Color("count()", title="Count").scale(scheme="greenblue"),
        )
    )
    return chart


entry_binding_corr_heatmap_1 = plot_entry_binding_corr(df_binding_effect_merge)
entry_binding_corr_heatmap_1.display()
if entry_binding_combined_corr_plot is not None:
    entry_binding_corr_heatmap_1.save(binding_corr_heatmap)
In [16]:
def plot_affinity_solid(df):
    df = df.dropna()
    # calculate r value
    slope, intercept, r_value, p_value, std_err = scipy.stats.linregress(
        df["binding_median_E2"], df["binding_median_E3"]
    )
    r_value = float(r_value)
    # make chart
    chart = (
        alt.Chart(
            df,
            title=alt.Title(
                "Correlation between Mutant Binding Scores", subtitle=f"r={r_value:.2f}"
            ),
        )
        .mark_point(color="black", size=30, opacity=0.2, filled=True)
        .encode(
            x=alt.X("binding_median_E2", title=("EFNB2 Binding")),
            y=alt.Y("binding_median_E3", title=("EFNB3 Binding")),
            tooltip=[
                "site",
                "wildtype",
                "mutant",
                "binding_median_E2",
                "binding_median_E3",
                "effect_E2",
                "effect_E3",
            ],
        )
    )
    min = int(df["binding_median_E2"].min())
    max = int(df["binding_median_E3"].max())
    text = (
        alt.Chart({"values": [{"x": min, "y": max, "text": f"r = {r_value:.2f}"}]})
        .mark_text(align="left", baseline="top", dx=-10, dy=-20)
        .encode(x=alt.X("x:Q"), y=alt.Y("y:Q"), text="text:N")
    )
    chart_and_text = chart
    return chart_and_text


E2_E3_corr = plot_affinity_solid(df_binding_effect_merge)
E2_E3_corr.display()
if entry_binding_combined_corr_plot is not None:
    E2_E3_corr.save(E2_E3_correlation)

Plot correlations between summary statistics for each site¶

In [17]:
def plot_affinity_solid_mean(df):
    df = df.dropna()
    means = (
        df.groupby("site")
        .agg(
            {
                "effect_E2": "median",
                "effect_E3": "median",
                "binding_median_E2": "median",
                "binding_median_E3": "median",
                "wildtype": "first",
            }
        )
        .reset_index()
    )
    slope, intercept, r_value, p_value, std_err = scipy.stats.linregress(
        means["binding_median_E2"], means["binding_median_E3"]
    )
    r_value = float(r_value)
    chart = (
        alt.Chart(
            means,
            title=alt.Title(
                "Correlation between Aggregate Mutant Binding Scores",
                subtitle=f"r={r_value:.2f}",
            ),
        )
        .mark_point(size=50, opacity=0.3)
        .encode(
            x=alt.X(
                "binding_median_E2",
                title=("Median EFNB2 Binding"),
                axis=alt.Axis(tickCount=3),
            ),
            y=alt.Y(
                "binding_median_E3",
                title=("Median EFNB3 Binding"),
                axis=alt.Axis(tickCount=3),
            ),
            tooltip=[
                "site",
                "wildtype",
                "binding_median_E2",
                "binding_median_E3",
                "effect_E2",
                "effect_E3",
            ],
        )
    )
    text = (
        alt.Chart({"values": [{"x": -3.5, "y": 0.5, "text": f"r = {r_value:.2f}"}]})
        .mark_text(align="left", baseline="top", dx=0, dy=-10)
        .encode(x=alt.X("x:Q"), y=alt.Y("y:Q"), text="text:N")
    )
    chart_and_text = chart
    return chart_and_text


E2_E3_site_corr = plot_affinity_solid_mean(df_binding_effect_merge)
E2_E3_site_corr.display()
if entry_binding_combined_corr_plot is not None:
    E2_E3_site_corr.save(E2_E3_correlation_site)

if entry_binding_combined_corr_plot is not None:
    (E2_E3_site_corr | E2_E3_corr).save(combined_E2_E3_site_corr)

Make plot showing binding by site (median)¶

In [18]:
def plot_affinity_by_site_median(df):
    variant_selector = alt.selection_point(
        on="mouseover", nearest=True, empty=False, fields=["site"], value=0
    )
    empty_charts = []
    for selection in ["binding_median_E2", "binding_median_E3"]:
        if selection == "binding_median_E2":
            name = "EFNB2 Binding"
        else:
            name = "EFNB3 Binding"
        mean = df.groupby("site")[selection].max().reset_index()
        mean = mean[mean[selection] >= 0]
        chart = (
            alt.Chart(mean)
            .mark_point(stroke="black", filled=True, size=50)
            .encode(
                x=alt.X(
                    "site",
                    title=("Site"),
                    axis=alt.Axis(grid=True, tickCount=4),
                    scale=alt.Scale(domain=[70, 602]),
                ),
                y=alt.Y(selection, title=(name), axis=alt.Axis(grid=True, tickCount=3)),
                tooltip=["site"],
                color=alt.condition(
                    variant_selector, alt.value("orange"), alt.value("black")
                ),
                opacity=alt.condition(variant_selector, alt.value(1), alt.value(0.5)),
                strokeWidth=alt.condition(variant_selector, alt.value(1), alt.value(0)),
            )
            .properties(height=150, width=500)
            .add_params(variant_selector)
        )
        empty_charts.append(chart)
    combined_chart = alt.vconcat(*empty_charts, spacing=1, title="Max Binding by Site")
    return combined_chart


binding_by_site = plot_affinity_by_site_median(df_binding_effect_merge)
binding_by_site.display()
if entry_binding_combined_corr_plot is not None:
    binding_by_site.save(binding_by_site_plot)
In [19]:
def plot_affinity_by_contact_site(df, sites_to_show, title_text):
    variant_selector = alt.selection_point(
        on="mouseover", nearest=True, empty=False, fields=["site"], value=0
    )
    empty_charts = []

    contact_df = df[df["site"].isin(sites_to_show)]
    sites = list(contact_df["site"].unique())

    for selection in df["selection"].unique():
        tmp_df = contact_df[contact_df["selection"] == selection]
        mean = tmp_df.groupby("site")["binding_median"].max().reset_index()

        chart = (
            alt.Chart(mean)
            .mark_point(size=100)
            .encode(
                x=alt.X(
                    "site:O",
                    sort=sites,
                    title=("Site"),
                    axis=alt.Axis(grid=True, labelAngle=-90),
                    scale=alt.Scale(domain=sites),
                ),
                y=alt.Y(
                    "binding_median", title=(f"{selection}"), axis=alt.Axis(grid=True)
                ),
                tooltip=["site"],
                color=alt.condition(
                    variant_selector, alt.value("orange"), alt.value("black")
                ),
                strokeWidth=alt.condition(variant_selector, alt.value(2), alt.value(0)),
            )
            .add_params(variant_selector)
        )
        empty_charts.append(chart)
    combined_chart = alt.vconcat(*empty_charts, spacing=1, title=title_text)
    return combined_chart


contact_binding_by_site = plot_affinity_by_contact_site(
    df_binding_effect_concat, config["contact_sites"], "Max Binding in Contact"
)
contact_binding_by_site.display()
if entry_binding_combined_corr_plot is not None:
    contact_binding_by_site.save(max_binding_in_contact)

contact_binding_by_site_stalk = plot_affinity_by_contact_site(
    df_binding_effect_concat, list(range(96, 147)), "Max Binding in Stalk"
)
contact_binding_by_site_stalk.display()
if entry_binding_combined_corr_plot is not None:
    contact_binding_by_site_stalk.save(max_binding_in_stalk)

Make bubble plots for binding in different areas of receptor pocket¶

In [20]:
def make_boxplot_binding_region(
    df, title
):  # Create a box plot using Altair for aggregated means
    barrel_ranges = {
        "Hydrophobic": config["hydrophobic"],
        "Salt Bridges": config["salt_bridges"],
        "Hydrogen Bonds": config["h_bond_total"],
        "Contact": config["contact_sites"],
        "Overall": list(range(71, 602)),
    }

    mean_df = df.groupby("site")[["binding_median"]].median().reset_index()
    custom_order = [
        "Hydrophobic",
        "Salt Bridges",
        "Hydrogen Bonds",
        "Contact",
        "Overall",
    ]
    agg_means = []

    # For each barrel, filter the site_means dataframe to the sites belonging to that barrel and then store the means
    for barrel, sites in barrel_ranges.items():
        subset = mean_df[mean_df["site"].isin(sites)]
        for _, row in subset.iterrows():
            agg_means.append(
                {"barrel": barrel, "effect": row["binding_median"], "site": row["site"]}
            )
        agg_means_df = pd.DataFrame(agg_means)
    chart = (
        alt.Chart(agg_means_df)
        .mark_point(size=50, opacity=0.4)
        .encode(
            x=alt.X(
                "barrel:O", sort=custom_order, title=None, axis=alt.Axis(labelAngle=-90)
            ),
            y=alt.Y(
                "effect",
                title=f"Median {title} Binding",
                axis=alt.Axis(grid=True, tickCount=4),
            ),
            xOffset="random:Q",
            tooltip=["barrel", "effect", "site"],
        )
        .transform_calculate(random="sqrt(-1*log(random()))*cos(2*PI*random())")
    )

    return chart.display()


make_boxplot_binding_region(df_E2_filter, "EFNB2")
make_boxplot_binding_region(df_E3_filter, "EFNB3")

make boxplot of binding scores by region¶

In [21]:
def make_boxplot_binding_region(df):
    barrel_ranges = {
        "Stalk": list(range(96, 147)),
        "Neck": list(range(148, 165)),
        "Linker": list(range(166, 177)),
        "Head": list(range(178, 602)),
        "Receptor Contact": config["contact_sites"],
        "Total": list(range(71, 602)),
    }
    custom_order = ["Stalk", "Neck", "Linker", "Head", "Receptor Contact", "Total"]
    empty_charts = []
    for selection in df["selection"].unique():
        tmp_df = df[df["selection"] == selection]
        agg_means = []

        # For each barrel, filter the site_means dataframe to the sites belonging to that barrel and then store the means
        for barrel, sites in barrel_ranges.items():
            subset = tmp_df[tmp_df["site"].isin(sites)]
            for _, row in subset.iterrows():
                agg_means.append(
                    {
                        "region": barrel,
                        "binding_median": row["binding_median"],
                        "site": row["site"],
                    }
                )
            agg_means_df = pd.DataFrame(agg_means)

        chart = (
            alt.Chart(agg_means_df, title=f"{selection}")
            .mark_boxplot(color="darkgray", extent="min-max", opacity=1)
            .encode(
                x=alt.X(
                    "region:O",
                    sort=custom_order,
                    title="RBP Region",
                    axis=alt.Axis(labelAngle=-90),
                ),
                y=alt.Y(
                    "binding_median",
                    title=f"Binding",
                    axis=alt.Axis(grid=True, tickCount=4),
                ),
                tooltip=["region", "binding_median", "site"],
            )
            .properties(width=config["width"], height=config["height"])
        )
        empty_charts.append(chart)
    combined_effect_chart = alt.hconcat(*empty_charts).resolve_scale(
        y="shared", x="shared", color="independent"
    )
    return combined_effect_chart


entry_region_boxplot = make_boxplot_binding_region(df_binding_effect_concat)
entry_region_boxplot.display()
if entry_binding_combined_corr_plot is not None:
    entry_region_boxplot.save(binding_region_boxplot_plot)
In [22]:
def make_bubble_binding_region(df):
    barrel_ranges = {
        "Stalk": list(range(96, 147)),
        "Neck": list(range(148, 165)),
        "Linker": list(range(166, 177)),
        "Head": list(range(178, 602)),
        "Receptor Contact": config["contact_sites"],
        "Total": list(range(71, 602)),
    }
    custom_order = ["Stalk", "Neck", "Linker", "Head", "Receptor Contact", "Total"]
    empty_charts = []
    for selection in df["selection"].unique():
        tmp_df = df[df["selection"] == selection]
        agg_means = []

        # For each barrel, filter the site_means dataframe to the sites belonging to that barrel and then store the means
        for barrel, sites in barrel_ranges.items():
            subset = tmp_df[tmp_df["site"].isin(sites)]
            for _, row in subset.iterrows():
                agg_means.append(
                    {
                        "region": barrel,
                        "binding_median": row["binding_median"],
                        "site": row["site"],
                        "mutant": row["mutant"],
                    }
                )
            agg_means_df = pd.DataFrame(agg_means)

        variant_selector = alt.selection_point(
            on="mouseover", empty=False, fields=["site", "mutant"], value=1
        )

        chart = (
            alt.Chart(agg_means_df, title=f"{selection}")
            .mark_point(opacity=0.3, stroke="black")
            .encode(
                x=alt.X(
                    "region:O",
                    sort=custom_order,
                    title="RBP Region",
                    axis=alt.Axis(labelAngle=-90),
                ),
                y=alt.Y(
                    "binding_median",
                    title=f"Binding",
                    axis=alt.Axis(grid=True, tickCount=4),
                ),
                xOffset="random:Q",
                tooltip=["region", "binding_median", "site", "mutant"],
                color=alt.condition(
                    variant_selector, alt.value("orange"), alt.value("black")
                ),
                opacity=alt.condition(variant_selector, alt.value(1), alt.value(0.1)),
                strokeWidth=alt.condition(variant_selector, alt.value(2), alt.value(0)),
                size=alt.condition(variant_selector, alt.value(50), alt.value(15)),
            )
            .transform_calculate(random="sqrt(-1*log(random()))*cos(2*PI*random())")
            .properties(width=config["width"], height=config["height"])
        ).add_params(variant_selector)
        empty_charts.append(chart)
    combined_effect_chart = (
        alt.hconcat(*empty_charts)
        .resolve_scale(y="shared", x="shared", color="independent")
        .add_params(variant_selector)
    )
    return combined_effect_chart


entry_region_bubble = make_bubble_binding_region(df_binding_effect_concat)
entry_region_bubble.display()
if entry_binding_combined_corr_plot is not None:
    entry_region_bubble.save(binding_region_bubble_plot)
In [ ]: